Medical Need: Human immunodeficiency virus (HIV), the etiologic agent of acquired immune-deficiency syndrome (AIDS), continues to infect millions of people worldwide. Despite recent improvements in anti-retroviral therapy, there remains an urgent need for novel and improved methods of treatment for individuals living with AIDS. Although highly active anti-retroviral therapy (HAART) has dramatically improved patient outcomes, the limitations of treatment options include the emergence of resistant viruses and deduced patient compliance as a result of adverse side effects. Therefore, the need for additional therapeutic modalities which are safe and convenient remains high. Interruption of the formation of the gp41 six-helical coiled coil, the final step of HIV-1 membrane fusion and infection of the host cell, is an example of a new treatment modality. Enfuvirtide (Fuzeon), recently approved by the FDA, is safe and effective in treating HIV-1 infection. Goal of Research: Locus Pharmaceuticals' innovative computational methodology, which requires only a crystal structure as input, rapidly and accurately identifies the active binding site of a protein and simultaneously enables design of novel, small molecule inhibitor compounds. This process was applied to the gp41 six-helical bundle crystal structure recently published by Chan and coworkers, resulting in the identification of a series of low molecular weight, drug-like molecules. The molecules interrupt HIV-1 gp41 six-helix bundle formation and protect cells in clinically relevant HIV-1 challenge assays. The focus of this proposal is to solve co-crystal structures of the compounds in complex with gp41. These structures will be used as input for further computational design and optimization of gp41 inhibitory molecules. Specific Aims: Produce and validate synthetic chimeric HR-1 peptide constructs and expressed HR-1 polypeptide constructs that retain the ability to form a six-helix bundle and have improved physicochemical properties that facilitate crystallization. Determine conditions under which the constructs form crystals.